GANP protein

ABSTRACT

The object of the present invention is to provide a novel protein having a kinase activity and a gene encoding said protein. According to the present invention, there is provided a GANP protein which is represented by the amino acid sequence shown in SEQ ID No. 1 or No. 3 of the sequence listing, and is involved in the signal conversion of abnormal B cell differentiation in an autoimmune state, and has a kinase activity, and a polynucleotide which encodes said protein.

TECHNICAL FIELD

The present invention relates to a novel protein having a kinaseactivity and a gene encoding said protein.

BACKGROUND ART

Antigen binding to the membrane lgR initiates the activation andmaturation of the antigen-specific B cells in the peripheral lymphoidorgans (Rajewsky, Nature (Lond.)., 381:751-758, 1996; Sakaguchi et al.,Adv. Immunol. 54:337-392, 1993). B cells enter the outer periarteriallymphoid sheath (PALS) (Rajewsky, Nature (Lond.)., 381:751-758, 1996)and initiate costimulus-dependent interactions with specific Th cellsand interdigitating dendritic cells within 48 h after immunization(MacLennan, Annu. Rev. Immunol. 12:117-139, 1994; Liu et al., Immunol.Rev. 156:111-126, 1997). Antigen-driven B cells proliferate in the outerPALS and then undergo further activation in the lymphoid follicles toestablish the germinal center (herein sometimes abbreviated as GC) (Hanet al., J. Immunol. 155:556-567, 1995; Jacob et al., J. Exp. Med.176:679-687, 1992; Kelsoe, Immunity 4:107-111, 1996). Such B cellsmature into large slg⁻ centroblasts that rapidly move through the cellcycle to form the dark zone and further mature into centrocytes thatexpress a unique surface character of PNA⁺B220⁺slgM⁺slgD⁻CD38⁻ in thelight zone of the GC (Kosco-Vilbois et al., 1997. Immunol. Today18:225-230, 1997; Kelsoe, Immunol. Today 16:324-326, 1995; Oliver etal., J. Immunol. 158:1108-1115, 1997).

Centrocytes presumably undergo the processes of either apoptosis oraffinity maturation of immunoglobulin V regions and the change processof class switching toward the lgG class antigen. Some centrocytessurvive for a longer period in the lymphoid compartment as memory Bcells. The other centrocytes probably migrate to the marginal zone ofthe GC and receive further antigenic stimulation and costimulatorysignals through B cell activation molecules, such as CD40 and CD38, andreceptors for various B cell stimulatory cytokines (Gray et al., J. Exp.Med., 180:141-155, 1994; Foy et al., J. Exp. Med., 180:157-163, 1994).Antigen-specific B cells further stimulated in this area probablymigrate into the interstitial region of the spleen (called red pulp),where various kinds of other immune-competent cells may interact withantigen-driven B cells. Histochemical analysis in several autoimmunemice identified unique antibody-producing cells in this area whichappear as plasma cells or aberrant plasma cells called Mott cells(Tarlinton et al., Eur. J. Immunol. 22:531-539, 1992; Jiang et al., J.Immunol., 158:992-997, 1997).

Autoimmunity is a phenomenon in which the impairment of self/nonselfdiscrimination occurs frequently in the antigen-specific lymphocytes(Theofilopoulos, Immunol. Today, 16:90-98, 1995). The immune systems ofvarious autoimmune diseases show the combinatory mechanism involving Tcells and B cells (Theofilopoulos et al., Adv. Immunol., 37:269-290,1985; Okamoto et al., J. Exp. Med. 175:71-79, 1992; Reininger et al., J.Exp. Med., 184:853-861, 1996; Theofilopoulos, et al., Immunol. Rev.55:179-216, 1981; Watanabe-Fukunaga et al., Nature (Lond.).,356:314-317, 1992; Takahashi et al., Cell, 76:969-976, 1994; Shlomchicket al., Nature (Lond.). 328:805-811, 1987).

NZB and NZW are the strains characterized by multiple genetic factorsgenerating the severe autoimmune state of SLE as (NZB×NZW)F₁ mice(Theofilopoulos et al., Adv. Immunol., 37:269-290, 1985; Okamoto et al.,J. Exp. Med., 175:71-79, 1992; Reininger et al., J. Exp. Med.,184:853-861, 1996; Theofilopoulos et al., Immunol. Rev., 55:179-216,1981). NZB mice spontaneously generate the state of autoimmunity withthe anti-red blood cell antibody that causes an autoimmune hemolyticanemia (Okamoto et al., J. Exp. Med., 175:71-79, 1992). NZW mice show aninsidious autoimmune phenomenon (Reininger et al., J. Exp. Med.184:853-861, 1996). The SLE state of (NZB×NZW)F₁ mice is apparentlycaused by multiple genetic factors associated with T and B cells(Theofilopoulos et al., Immunol. Rev., 55:179-216, 1981). NZB mice showan apparent abnormality of B cells, but the molecular mechanism of theabnormal B cell activation in NZB mice remains to be elucidated.

DISCLOSURE OF THE INVENTION

To address the issue of which molecules are involved in such maturationof B cells, the present inventors prepared monoclonal antibodies againstintracellular components of a murine B cell line WEHI-231, which has theNZB genetic background. A monoclonal antibody named 29-15 recognizes adifferentiation antigen whose expression is augmented in GC-B cells ofperipheral lymphoid organs. With the 29-15 monoclonal antibody, thepresent inventors studied the expression of the antigen in peripherallymphoid organs, which characterized the molecule as a differentiationantigen upregulated in the light zone of the GC from hyperimmunizedmice. In the spleen of NZB mice, lgM-producing plasma cells with highexpression of the GANP antigen appear before the onset of autoimmunity,which would suggest that this is an important molecular event forunderstanding the peripheral immune response and autoimmunity withautoantibodies.

The present inventors have studied to identify the above-mentionedantigen whose expression is selectively increased in centrocytes ofgerminal center, and confirmed by in situ RNA hybridization using anisolated cDNA probe (ganp probe) that the expression of ganp mRNA isincreased in the area stained with 29-15 monoclonal antibody. It wasalso confirmed that the gene product, GANP protein, is a protein of 210kD which is localized in cytoplasma and nucleus, and is structurallysimilar with a transcription regulating factor in yeasts, SAC3. When Bcells are activated with anti-IgM antibody and anti-CD40 antibody, theamount of kinase which binds to GANP protein increased. These resultssuggests that GANP protein may be involved in a signal conversion ofabnormal B cell differentiation in certain autoimmune state. The presentinvention has been completed on the basis of these findings.

Thus, the present invention provides a GANP protein represented by theamino acid sequence shown in SEQ ID No.1 or No.3 of the sequencelisting. According to the present invention, there is provided a GANPmutant protein which is consisted of the amino acid sequence wherein oneor more amino acids are deleted, one or more amino acids are substitutedwith other amino acid(s), and/or one or more other amino acids are addedin the amino acid sequences shown in SEQ ID No.1 or No.3 of the sequencelisting, and has a kinase activity similar with that of GANP protein.According to the present invention, there is provided a polypeptidewhich contains, as a partial sequence, a full length amino acid sequenceof the aforementioned GANP protein or the aforementioned GANP mutantprotein.

According to another aspect, the present invention provides apolynucleotide which encodes the aforementioned GANP protein or GANPmutant protein. The typical polynucleotide is DNA encoding GANP proteinderived from mammal, and the DNA of mammal gene is preferred among them.Examples of most preferred polynucleotide are represented by the basesequences shown in SEQ ID No. 2 (DNA sequence encoding GANP protein frommouse) or SEQ ID No. 4 (DNA sequence encoding GANP protein from human)of the sequence listing.

Further, according to the present invention, there is provided anantisense polynucleotide which is composed of the base sequence of anantisense chain of the aforementioned polynucleotide, or derivatives ofsaid antisense polynucleotide. Furthermore, according to the presentinvention, there is provided a polynucleotide or antisensepolynucleotide of continuous 12 or more bases which is a partialsequence of the aforementioned polynucleotide or the aforementionedantisense polynucleotide, and a chemically modified polynucleotide orantisense polynucleotide of the aforementioned polynucleotide or theaforementioned antisense polynucleotide.

According to further another aspect, the present invention provides amethod for obtaining DNA of the base sequence shown in SEQ ID No. 2 orNo. 4 of the sequence listing or DNA which is the homologue from othermammal, wherein the aforementioned polynucleotide or antisensepolynucleotide is used as a probe, and cDNA which hybridizes to theprobe is obtained from mammal cDNA library. The length of the cDNA isalmost the same as that of GANP gene, and the protein encoded by it hasapproximately 210 kDa. Further, according to the present invention,there is provided cDNA obtained by the aforementioned method and GANPprotein encoded by it.

According to further another aspect of the present invention, there isprovided an antibody which recognizes GANP protein or GANP mutantprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing detection of 29-15⁺cells in the PP ofnormal mice. The immunohistochemical analysis was carried out on PP withthe 29-15 mAb and ALP-anti-rat lg antibody. Positive cells appear in thecentral area with Vector Blue ALP substrate, and the strong signal inthe surrounding area is in intestinal villi containing nonspecificendogenous ALP activity. For two-color staining, the sections arefurther stained either with biotin-anti-B220 mAb or biotin-anti-lgD mAbfollowed by HRP-streptavidin in combination with DAB.

FIG. 2 is a photograph showing appearance of 29-15⁺ cells in the GC areaof SRBC-immunized mice. Normal BALB/c mice were injected four times withSRBC during 12 days and the spleen sections were stained withhematoxylin or studied by immunohistochemistry as in FIG. 1. Thesections of normal and SRBC-immunized BALB/c mice are parallel, whencompared after staining with the 29-15 mAb.

FIG. 3 is a photograph showing appearance of 29-15⁺ cells in the GC areaof SRBC-immunized mice. The sections of the GC area are stained withPNA, anti-BrdU, and the 29-15 mAb in combination with the individualcolors as described in the Materials and Methods. Upper photograph showshematoxylin staining of the GC area (GC) and the central artery (CA).Middle photograph shows three-color staining, indicating the 29-15⁺PNA⁺cells. Lower panel shows a schema of 29-15⁺PNA⁺ cells.

FIG. 4 is a photograph showing expression of the GANP^(dense+) cells inthe red pulp area of autoimmune-prone mice. Sections were prepared fromthe spleens of nonimmunized mice of BALB/c, NOD, NZB, (NZB×NZW)F₁, BXSB,and MRL/lpr. All mice were used 6-8 weeks after birth. The GANP^(dense+)cells stained with the 29-15 mAb appear in the red pulp area of NZB,(NZB×NZW)F₁, MRL/lpr, and BXSB strains.

FIG. 5 is a photograph showing expression of the GANP^(dense+) cells inthe red pulp area of autoimmune-prone mice. Sections of the LN ofpopliteal regions were stained with the 29-15 mAb. The GANP^(dense+)cells appear in peripheral LN of older NZB mice (10 month old) andMRL/lpr mice (8 week old).

FIG. 6 is a photograph showing characterization of the GANP^(dense+)cells in the autoimmune-prone mice. Sections were prepared with thespleen of nonimmunized NZB mice (8 week old). Immunohistochemicalanalysis was performed with the 29-15 mAb in combination with one of thefollowing reagents: anti-B220, PNA.

FIG. 7 is a photograph showing characterization of the GANP^(dense+)cells in the autoimmune-prone mice. Sections were prepared with thespleen of nonimmunized NZB mice (8 week old). Immunohistochemicalanalysis was performed with the 29-15 mAb in combination with one of thefollowing reagents: anti-lgM, anti-Syndecan-1, or anti-BrdU mAb.

FIG. 8 is a photograph showing Mott cells that appear in NZB mice by PASstaining.

FIG. 9 is a diagram showing a deduced amino acid sequence of mouse GANPprotein in one character notation.

FIG. 10 is a diagram showing a structure of the GANP protein. In thefigure, S/T rich region: serine/threonine rich region, SAC3 homologyregion; SAC3 homology region, nuclear localizing signal: nuclearlocalizing signal. Four LXXLL motifs are present.

FIG. 11 is a photograph showing a result of in situ RNA hybridization ofthe ganp gene. Sections of spleens from SRBC-immunized, nonimmunizedBALB/c, and NZB mice were hybridized with the ganp anti-sense probe. Inthe figure, the white pulp area (WP), red pulp area (RP), and GC area(GC) are indicated. The GANP^(dense+) cells were recognized in the redpulp of NZB mice.

FIG. 12 is a diagram showing the results of the analysis by Westernblotting after immunoprecipitation of GANP protein. The GANP protein wasdetected as a 210-kD protein expressed in cytoplasmic and nuclearfractions of WEHI-231 cells.

FIG. 13 is a diagram showing the results where spleen B cells fromnormal BALB/c mice were stimulated with F(ab′)₂ of goat anti-lgM Ab (10μg/ml) and anti-CD40 mAb (10 μg/ml) for 48 hour and stained with theanti-GANP mAb.

FIG. 14 is a diagram showing the results where in vitro kinase reactionwas carried out with the anti-GANP immunoprecipitates in the presence of[γ-³²P]-ATP for 10 minutes. Phosphorylation on the proteins weredetected by the autoradiography after SDS-PAGE separation.Phosphorylation of the GANP is indicated with an arrow (Figure A), andphosphoamino acid analysis of phosphorylated GANP protein is also shown(Figure B).

FIG. 15 is a diagram of the structure of the mouse GANP protein. In thefigure, the homologous region to SAC3 and Map80, nuclear localizationsequences (NLSs), and coiled-coil regions are indicated. Four LXXLLmotifs are indicated by black.

FIG. 16 shows a result of RT-PCR assay. The upregulation of gnap mRNA inanti-μ- and anti-CD40-stimulated B calls in vitro is shown. HPRT wasused as a control to confirm the amount of each template.

FIG. 17 shows a result of in vitro kinase reaction. The call lysate wasprepared from unstimulated (left) or stimulated (right) cells andsubjected to anti-GNAP immunoprecipitation. In vitro kinase reaction wascarried out with the anti-GNAP (42-23) immunoprecipitates in thepresence of [γ-³²P]-ATP for 10 minutes. Phosphorylation on the proteinswas detected by the autoradiography after SDS-PAGE separation. An arrowindicates the position of phosphorylated GNAP.

FIG. 18 is a scheme showing a physical association between GNAP andMCM3. The cell lysate from WEHI-231 was immunoprecipitated withanti-GST, anti-GNAP (42-23), or anti-MCM3 Ab. After separation bySDS-PAGE, the proteins were electrophoretically transferred to amembrane and probed with anti-MCM3 Ab.

FIG. 19 is a scheme showing a physical association between GNAP andMCM3. Anti-GST, anti-GNAP (42-23) and anti-MCM3 immunoprecipitates fromWEHI-231 cell lysates were subjected to in vitro kinase assay. Normalrabbit serum (NRS) was used as a control for anti-MCM3 Ab. The sampleswere separated by 7% SDS-PAGE. The bands corresponding to GNAP and MCM3were indicated by arrows in the left panel. On the right panel, V8cleavage mapping of 210-kDa bands showed an identical cleavage pattern.As a control an irrelevant V8-digested protein was separated inparallel.

FIG. 20 is a scheme showing a result where double staining withanti-MCM3 Ab and anti-CR1 mAb, or PNA, was performed. The expression ofMCM3 was upregulated in GC area.

FIG. 21 is a scheme where a deduced amino acid sequence of human GANPprotein is represented in one character notation.

FIG. 22 is a photograph showing a result where human ganp and Map 80were mapped by FISH method using human chromosome.

PREFERRED EMBODIMENT FOR CARRYING OUT THE INVENTION

The typical examples of GANP protein of the present invention areprotein represented by the amino acid sequences of SEQ ID No. 1 and No.3 of the sequence listing, and are characterized in that they have amolecular weight of 210 kDa and have a kinase activity. GANP mutantproteins provided by the present invention are represented by the aminoacid sequences wherein approximately 1 to several preferably 1 to 20,more preferably 1 to 10, most preferably 1 to 5 amino acid residues aresubstituted, inserted, and/or deleted in the amino acid sequences of SEQID No. 1 or No. 3, and have a kinase activity which is substantiallysimilar with GANP protein represented by the amino acid sequences of SEQID No. 1 or No. 2. These GANP mutant proteins are within the scope ofthe present invention. The protein represented by the amino acidsequences of SEQ ID No. 1 or No. 3 of the sequence listing and homologuethereof are those whose expression is selectively increased incentrocytes of germinal center of mammal from which the protein isderived.

Usually, the active domain of GANP protein or GANP mutant protein can bereadily identified by preparing a polypeptide wherein amino acidresidue(s) are deleted from N-terminal and/or C-terminal of the fulllength amino acid sequence, and measuring the kinase activity of thepolypeptide. The polypeptides provided by the present invention arethose comprised of an active domain of GANP protein and GANP mutantprotein and those comprising, as a partial sequence, a polypeptidecomprised of said active domain, and have a kinase activity which issubstantially similar with GANP protein. Moreover, another polypeptidesprovided by the present invention are those comprising, as a partialsequence, a full length amino acid sequence of GANP protein or GANPmutant protein, and have a kinase activity which is substantiallysimilar with GANP protein.

The polynucleotide provided by the present invention includes DNA andRNA as well as all of the nucleotides obtained by chemically modifyingDNA or RNA. The term “polynucleotide” used herein should be most broadlyinterpreted to include non-naturally occurring form. The typicalexamples of the polynucleotide provided by the present invention are DNAor RNA which encodes the aforementioned GANP protein or GANP mutantprotein. Another example of the polynucleotide of the present inventionis antisense polynucleotide.

It is well known for a skilled person in the art that, using degeneracyof genetic code, at least partial bases of a polynucleotide can bereplaced with another type of bases without changing the amino acidsequence of the polypeptide which is produced from the polynucleotide.Therefore, the polynucleotide of the present invention includes allpolynucleotides which encode GANP protein or GANP mutant protein. Asexamples of the preferred gene of the present invention, a gene encodingGANP protein from mouse is shown in SEQ ID No.2 of the sequence listing,and a gene encoding GANP protein from human is shown in SEQ ID No.4. Theamino acid sequence of GANP mutant protein can be determined from thebase sequence of a gene encoding said mutant. For example, sequencingcan be carried out by using commercially available programs (forexample, MacVector®, Eastman Chemical), or Genetic (Software Kaihatsu)).

The scope of the present invention covers antisense polynucleotidescomposed of a base sequence of antisense chain of polynucleotideencoding GANP protein, and derivatives thereof. The antisensepolynucleotides is provided as an embodiment of the polynucleotidementioned above, and the term “antisense polynucleotide” may be hereinused to clearly mean that it is a polynucleotide comprised of basesequence of antisense chain. The antisense polynucleotide can hybridizeto polynucleotide encoding GANP protein, and if the polynucleotide towhich it hybridize is a polynucleotide of coding region, thebiosynthesis of the polypeptide encoded by the polynucleotide can beinhibited.

Antisense polynucleotide for inhibiting the biosynthesis of polypeptidepreferably contains 12 or more bases. On the other hand, anunnecessarily long sequence is not preferred in order to incorporatefull length antisense polynucleotide into cells. When an antisensepolynucleotide is incorporated into cells to inhibit the biosynthesis ofGANP protein, it is preferred to use an antisense polynucleotide of 12to 30 bases, preferably 15 to 25 bases, more preferably 18 to 22 bases.

The antisense polynucleotide of the present invention or derivativesthereof include all of the form where several nucleotides composed ofbase, phosphoric acid and sugar are bound whether or not they arepresent in nature. Typical examples include a naturally occurringantisense DNA and antisense RNA. Non-naturally occurring polynucleotidesinclude, for example, polynucleotides of methylphosphonate type andphosphorothioate type. As to the antisense polynucleotide of the presentinvention, various antisense polynucleotide derivatives which areexcellent in binding ability to target DNA or mRNA, tissueselectability, cell permeation property, nuclease resistance,intercellular stability and the like, can be obtained by using anantisense technology available to a skilled person in the art.

Generally, in view of easiness of hybridization, it is preferred todesign an antisense polynucleotide or derivatives thereof having a basesequence complementary with base sequence which forms a loop of RNA.Therefore, as to the antisense polynucleotide of the present inventionand derivatives thereof those which hybridize to loop region of RNA arepreferred examples. Moreover, an antisense polynucleotide having asequence complementary with a translation initiation codon andneighborhood thereof ribosome binding site, capping site, or splicingsite can generally be expected to exhibit high expression inhibitioneffect. Therefore, the antisense polynucleotide of the present inventionor derivatives thereof having a sequence complementary with atranslation initiation codon and neighborhood thereof, ribosome bindingsite, capping site, and/or splicing site of the gene encoding GANPprotein are preferred example in view of expression inhibition effect.

Among the currently generally known polynucleotide derivatives, thederivatives where at least one of nuclease resistance, tissueselectability, cell permeation property and binding ability is enhanced,preferably include derivatives having a phosphorothioate bond as askeleton structure. The polynucleotide of the present invention andderivatives thereof include derivatives having these function orstructure.

Among the antisense polynucleotide of the present invention, naturallyoccurring type of antisense polynucleotide may be synthesized by using achemical synthesizer or may be prepared by a PCR method using a DNAencoding GANP protein as a templeta. Polynucleotide derivatives such asmethylphosphonate type or phosphorotioate type may usually be preparedby chemical synthesis. In this case, the procedure can be carried outaccording to an instruction attached with the chemical synthesizer, andthe synthesized product thus obtained-can be purified by HPLC methodusing reverse phase chromatography and the like.

Polynucleotide which is a polynucleotide encoding GANP protein of thepresent invention, antisense polynucleotide thereof, or a portionthereof (for example, polunucleotide composed of continuous 12 or morebases) can be used as a probe for screening a DNA encoding GANP proteinfrom mammalian cDNA library. For such a purpose, a polynucleotidecomposed of a sequence of continuous 15 or more bases is particularlypreferred. The polynucleotide used as a probe may be a derivative.Usually, it is recognized that a sequence having the aforementionednumber or more of base is a specific sequence.

A DNA of continuous 12 or more bases in the base sequence of SEQ ID No.2 or No. 4 of the sequence listing, or a polynucleotide which hybridizesto said DNA (antisense polynucleotide) can be used as a probe forscreening a DNA encoding GANP protein from cDNA library or the like.

Also, a tissue which expresses mRNA from GANP gene can be detected byperforming a Northern Blot hybridization on mRNA derived from varioustissues by using a polynucleotide encoding GANP protein of the presentinvention, antisense polynucleotide thereof, or a polynucleotide of aportion thereof as a probe. Furthermore, a polynucleotide of 12 or morebases can be used as a primer for polymerase chain reaction (PCR), and apolynucleotide encoding GANP protein can be obtained by PCR. Also, theprimer can be appropriately selected to clone any portion of GANPprotein.

As to the cDNA library used in the screening using the aforementionedprobe, one prepared from mRNA can be preferably used. A group of cDNAselected by random sampling from these cDNA library may be used as asample for screening. A commercially available cDNA library can be used.

The cDNA which hybridizes to the above-obtained GANP gene is insertedinto a suitable vector (for example, pGEX-4T-1 vector), and isintroduced into a host (for example, E. coli) to prepare a transformant.The type of the vector and the type of the host are not particularlylimited, and any suitable expression vector may be selected and useddepending on the type of the host. As the host, bacterium such as E.coli, yeasts, or animal cells can be used. A method for obtaining atransformant by introducing a recombinant vector into a suitable hostsuch as E. coli is not particularly limited, and any method available toa skilled person in the art may be applied.

The transformant into which the GANP gene of the present invention wasintroduced can be cultured to amplify a gene DNA or produce a protein,thereby prodicing GANP protein. The preparation and culturing of atransformant are described in various literatures and reports, and manymethods have been developed and have been conventionally used in theart. Therefore, a skilled person in the art can easily prepare GANPprotein on the basis of the base sequence described herein. The methodsfor introducing a gene into cells include calcium chloride method,lipofection method, protoplast method, and electroporation method.

Separation and purification of a protein of interest from the culturecan be carried out by using any means available to a skilled person inthe art in combination appropriately. For example, GANP protein of thepresent invention can be efficiently recovered and purified byperforming procedures such as concentration, solubilization, dialysis,various chromatography and the like. More specifically, selection may besuitably made among immunoprecipitation, salting out, ultrafilteration,isoelectric point precipitation, gel filteration, electrophoresis,various chromatography such as ion exchange chromatography, hydrophobicchromatography and antibody chromatography, chromatofocusing, adsorptionchromatography, and reverse phase chromatography. By using a geneencoding GANP mutant protein, GANP mutant protein can be similarlyprepared.

Also, GANP protein or GANP mutant protein can be prepared as a fusedprotein with another polypeptide. Such a fused polypeptide is within thescope of the present invention. The type of the polypeptide to be fusedis not particularly limited, and includes, for example, a signal peptidewhich promotes an extracellular secretion. The preparation of such afused protein may be carried out by using transformant. When a fusedprotein is used to prepare GANP protein or GANP mutant protein, a fusedprotein is treated with a chemical substance such as bromecyan or anenzyme such as protease, and the substance of interest which was cut outmay be separated and purified.

Antibodies which recognize GANP protein or GANP mutant protein can beprepared by using GANP protein or GANP mutant protein of the presentinvention or partial polypeptide thereof. The antibody of the presentinvention can be prepared by any means of a conventional method in theart by immunizing a mammal with GANP protein or GANP mutation protein.It can be confirmed by Western blotting, ELISA, immunostaining (forexample, measurement with FACS) or the like that the antibody recognizesGANP protein or GANP mutation protein of the present invention. Asimmunogens, there may used GANP protein or GANP mutant protein as wellas a portion thereof bound to another carrier protein such as calf serumalbumin. A portion of GANP protein or GANP mutation protein preferablycontains 8 or more amino acid residues, and such a polypeptide may besynthesized by using, for example, a peptide synthesizer.

A monoclonal antibody which is produced from hybridoma prepared by usinglymphocytes of immunized animals may be used as an antibody of thepresent invention. The process for the preparation of a monoclonalantibody is well known in the art and is conventionally used(“Antibodies, A Laboratory Manual” (Cold Spring Harbor Laboratory Press,1988), Chapter 6). Moreover, a fragment of antibody having aantigen-antibody reaction activity and a chimera antibody may be used asan antibody of the present invention. GANP protein or GANP mutantprotein of the present invention can be detected by a method using anantibody or a method using an antibody and an enzyme.

The present invention is illustrated in detail by the examples below,but the scope of the present invention is not limited to the examplesbelow.

EXAMPLE Example 1 Cloning of Mouse GANP Gene and Analysis of Expression

<Materials and Methods>

(1) Animals and Immunization

BALB/c mice and Lewis rats were purchased from Seac Yoshitomi Ltd.(Fukuoka). NZB, NZW, (NZB×NZW)F₁ mice (7 week old, female), MRL/lpr mice(8 week old, female), and BXSB mice (7 week old, male) were obtainedfrom Japan SLC Co. (Shizuoka). Aged NZB mice (10 month old, female) werekindly gifted from Dr. Sachiko Hirose (Department of Pathology, JuntendoUniversity School of Medicine). NOD mice (7 week old, male) weregenerously provided from Dr. Junichi. Miyazaki (Department of Nutritionand Physiological Chemistry, Osaka University Medical School). Allanimals were maintained in Center for Animal Resources and Developmentin Kumamoto University. BALB/c mice were immunized multiply with sheepred blood cells (Nippon Bio-Test Laboratories, Inc., Tokyo). Theimmunization was performed intravenously with 5-day interval andsections of the thymus, spleen, lymph node (LN), and Peyer's patches(PP) were prepared for the immunohistochernical analysis.

(2) Cells and Cell Culture

Splenic B cells from BALB/c mice were enriched as described previously(Nomura et al., Immunol. Lett. 45:195-203, 1995). These cells werecultured in RPM1-1640 medium (Gibco-BRL, Gaithersburg, Germany)containing 10% heat-inactivated FCS (Dainippon Pharmaceutical Co.,Osaka, Japan), 5 mM L-glutamine (Biowhitteker, Walkersville, Md., USA),100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-ME at 37° C. inan incubator with 5% carbon dioxide.

(3) Establishment of the 29-15 Monoclonal Antibody (Hereinafter Referredto as “25-15 mAb”)

The mAbs against a murine B cell line WEHI-231, which was establishedfrom a (BALB/c×NZB)F₁ mouse with mineral oil, were prepared by themethod described previously (Kuwahara et al., J. Immunol. 152:2742-2752,1994). Briefly, the cell lysate of WEHI-231 with the surface phenotypeslgM⁺slgD⁺B220⁺ was prepared with the hypotonic buffer in the absence ofdetergent and dialyzed against a phosphate buffered saline (PBS) inaccordance with the method of Sakaguchi et al (Sakaguchi et al., EMBO(Eur. Mol. Biol. Organ.) J. 5:2139-2147, 1986). The cell lysate wasimmunized into the foot pads of Lewis rats in the complete Freund'sadjuvant (CFA) (Difco Laboratories, Detroit, Mich., USA) and boostedtwice in the incomplete Freund's adjuvant (IFA) (Difco Laboratories) atday 4 and day 8. After 9 days, the lymph node of popliteal and inguinalregions were excised and the lymphoid cell suspension was prepared.Establishment of hybridomas, selection in the HAT media (Gibco-BRL), andrecloning of hybridoma clones were performed as described previously(Kuwahara et al., J. Immunol. 152:2742-2752, 1994). The 29-15 mAb wasselected to stain lymphoid cells in the immunohistochemical analysis.

(4) Antibodies and Reagents

F(ab′)₂ fragment of the affinity-purified goat anti-mouse μ antibody(ICN Pharmaceutical, Inc., Costa Mesa, Calif., USA), biotin-conjugatedpeanut agglutinin (PNA) (Vector Laboratories, Inc., Burlingame, Calif.,USA), biotin-conjugated anti-CD35 mAb (PharMingen, San Diego, Calif.),alkalinephosphatase (ALP) conjugated goat anti-rat IgAb (#59301, ICN),HRP conjugated goat anti-rat IgAb (ICN), HRP conjugated streptavidin(Kirkegaard & Perry Laboratories, Inc., Gaitherburg, Md.), ALPconjugated goat anti-mouse IgAb (Sigma Chemicals Co., St. Louis, Mo.),FITC conjugated mouse anti-rat κ mAb (ICN), PE conjugated anti-B220 mAb(PhaMingen), and ALP conjugated goat anti-rabbit IgAb (ZymedLaboratories Inc., South San Francisco, Calif.) were purchased and used.Biotin-conjugated mAbs such as anti-B220 (RA3-6B2), anti-μ (AM/3), andanti-δ (CS/15) were prepared in our laboratory. Anti-CD40 mAb (LB429)was established in our laboratory (Nomura et al., Immunol. Lett.45:195-203, 1995). Hybridomas of AM/3 and CS/15 were kindly provided byDr. Kensuke Miyake (Department of Immunology, Saga Medical School).Biotin-conjugated anti-Syndecan-1 was purchased from PharMingen (SanDiego, Calif., USA). Anti-BrdU mAb was obtained from NovocastraLaboratories, Ltd. (Newcastle, United Kingdom). Rabit anti-mouse MCM3/P1Ab is described in the literature(Kimura, H et al, 1994, EMBO J. 13,4311-4320).

(5) Immunohistochemistry

Immunohistochemical staining was performed as described previously(Ezaki et al., Arch. Histol. Cytol. 58:104-115, 1995; Yamanouchi et al.,Eur. J. Immunol. 28:696-707, 1998). In brief, the target organs excisedfrom BALB/c, NZB, (NZB×NZW)F₁, NOD, BXSB, and MRL/lpr mice were placedin OCT compound (Miles Inc., Elkhart, Ind., USA). The 6-μm cryosectionsplaced on the gelatin-coated slides were air-dried fully. The slideswere then fixed in acetone for 10 minutes, followed by rehydration inPBS for 15 minutes. The slides were incubated with the 29-15 mAb for 60minutes and were washed with PBS several times. After incubation withalkaline phosphatase-conjugated goat anti-rat lg antibody (ALP-anti-ratlg, catalogue #59301, ICN Pharmaceutical, Inc.), the slides were washedfour times with PBS. The slides were developed using Vector Blue (VectorLaboratories).

For secondary staining, the slides were incubated with biotin-labeledmAbs in combination with horseradish peroxidase (HRP)-conjugatedstreptavidin (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.,USA). After development with p-dimehylaminoazobenzene (DAB, Dojindo,Kumamoto), the sections were fixed lightly with 1% glutaraldehydesolution in PBS. To detect the cells of active proliferation in vivo,BrdU (Sigma Chemicals Co., St. Louis, Mo., USA) was injectedintravenously 1 hour before obtaining the organs. Cells undergoing DNAsynthesis were detected by staining with anti-BrdU mAb in combinationwith ALP-conjugated goat anti-mouse lg Ab (Sigma Chemicals Co.) followedby development with Vector Red (Matsuno et al., Cell Tissue Res.257:459-470, 1989). Periodic acid Schiff (PAS) staining was performed asdescribed previously (Jiang et al., J. Immunol. 158:992-997, 1997). Allsections were mounted by Aquatex (E. Merck, Darmstadt, Germany).

(6) Molecular Cloning of the cDNA Using λgt11 Vector

The cDNA libraries constructed with mRNAs from the mouse spleen, mousebone marrow, WEHI-231 cells and A20 cells were screened with thesupernatant of the 29-15 mAb after transferring the fusion protein ontonitrocellulose filters (Schleicher and Schuell, Darmstadt, Germany) thatwere presoaked with 20 mM IPTG (Inui et al., J. Immunol. 154:2714-2723,1995). The phage plates were incubated for 4 hours at 42° C. and thenthe plates were covered with the filters and further incubated for 4hours at 37° C. The filters were washed three times with the washingbuffer (PBS containing 0.1% Tween 20), blocked for 1 hour in theblocking buffer (5% nonfat dry milk in PBS containing 0.1% Tween 20),and then incubated with the 29-15 mAb. Positive signals were detected byautoradiography using ¹²⁵l-labeled sheep anti-rat lg Ab (Amersham,Buckinghamshire, United Kingdom). The initial cDNA clone contained a280-bp fragment that is capable of coding a polypeptide as a fusionprotein. With the original 280-bp fragment, the longer cDNA clones wereisolated from another WEHI-231 cDNA library. The 4.9-kb fragment of thesecond cDNA clone encodes a longest open reading frame of 4.5 kb. Tofurther determine the 5′ sequence, the 5′-RACE method was employed. Therace kit of Gibco-BRL was used.

(7) In situ RNA Hybridization on Tissue Sections

In situ RNA hybridization was carried out as described previously (Kondoet al., Blood 80:2044-2051, 1992). Paraffin-embedded sections weremounted on silanized slides. After the slides were deparaffinized,hybridization with ganp 280-bp riboprobe labeled by digoxigenin wasperformed for 16 hours at 50° C. The slides were washed with TNE buffer(10 mM Tris-HCl [pH 7.6], 500 mM NaCl, 1 mM EDTA) at 37° C. severaltimes, followed by washing with 2× and/or 0.2×SSC solution at 50° C.While using anti-digoxigenin antibody, the development was performed inthe presence of ALP substrate.

(8) Preparation of GST-cDNA Fusion Protein and Another Anti-GANP mAb

The ganp cDNA fragment encoding a part of GANP (amino acids of 679th to1028th of the amino acid sequence of SEQ ID No. 1 of the sequencelisting) was introduced into a pGEX-4T-1 vector (Pharmacia Biotech,Piscataway, N.J., USA). The recombinant plasmid was verified by DNAsequencing of the entire insert and the junction. The GST-GANP fusionprotein was prepared by glutathione-Sepharose (Pharmacia) columnchromatography as described elsewhere (Inui et al., J. Immunol.154:2714-2723, 1995). Anti-GANP mAb, designated 42-23, was establishedby immunizing the fusion protein in rats as described above.

(9) Western Blot Analysis

Protein gel electrophoresis, Western blot transfer, and theimmunodetection of proteins were performed as described previously(Kuwahara et al., Int. Imnmunol. 8:1273-1285, 1996). Fifty million cellswere lysed with 1 ml of the TNE lysis buffer (10 mM Tris-HCl [pH 7.8],150 mM NaCl 1 mM EDTA, 1% NP-40, 0.02% NaN₃) and the immune complex wasanalyzed on SDS-PAGE (7%). After the proteins were transferred onto anitrocellulose filter, the filter was blocked with PBS-Tween 20containing 5% nonfat dry milk and incubated with anti-GANP mAb for 60minutes. After washing with PBS-Tween 20 several times, the filter wasincubated with HRP-conjugated goat anti-rat lg (ICN Pharmaceutical,Inc.) for 30 minutes. The development was performed using an ECLdetection kit (Amersham).

(10) Subcellular Fractionation

Separation of intact nuclei was carried out as described previously(Schriber et al., Nucleic Acids Res. 17:6419, 1989). WEHI-231 cells werewashed with TBS and the pellets were resuspended in buffer A (10 mMHEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mMPMSF) and incubated for 15 minutes on ice, followed by the addition ofNP-40 to a final 1%. After the centrifugation, the supernatants wererecovered as a cytoplasmic fraction. The pellets were resuspended withthe same buffer and homogenized to obtain the intact nuclei by staining.The sample was centrifuged and the pellet was resuspended with coldbuffer C (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMDTT, 1 mM PMSF) and centrifuged. The supernatants were frozen at −80° C.as a nuclear fraction.

(11) In Vitro Kinase Reaction and Phosphoamnino Acid Analysis

Kinase reaction was carried out in vitro with the immunoprecipitate asdescribed previously (Kuwahara et al., J. Immunol. 152:2742-2752, 1994).Splenic B cells were purified by the method described (Nomura et al.,Immunol. Lett. 45:195-203, 1995). The B cells were stimulated in vitrofor 48 hours with F(ab′)₂ fraction of goat anti-lgM Ab and anti-CD40 mAb(LB429) as described previously (Nomura et al., Immunol. Lett.45:195-203, 1995). After harvesting and washing, cells were lysed withTNE Iysis buffer and immunoprecipitated with the anti-GANP mAb (42-23).The immunoprecipitates were incubated with [γ-³²p]-ATP (Amersham) andthe radiolabeled proteins were analyzed on SDS-PAGE (7%) and withautoradiography. The band corresponding to GANP was excised from driedgel. After SDS was removed from the gel, the homogenized gel wasdigested by TPCK-trypsin (Sigma Chemicals Co.) at 37° C. overnight. Thesamples were subjected to hydrolysis with 6N HCl and electrophoresedonto TLC (E. Merck).

V8 cleavage mapping of the indicated proteins was carried out asdescribed previously (Kuwahara, K., et al, 1994, J. Immunol.152:2742-2752).

(12) Cytoplasmic Staining

The cells were fixed with 2.5% paraformaldehyde solution in PBS followedby permeabilization with 70% ethanol for 1 hour on ice. The cells wereincubated with the 29-15 mAb in combination with FITC-conjugated mouseanti-rat κ mAb. Antibody-binding was analyzed on FACScan flow cytometer(Becton-Dickinson, Mountain View, Calif., USA).

(13) Immunoprecipitation and Western blot Analysis

Proteins obtained in the aforementioned (10) subcellular fractionationwere immunoprecipitated with the anti-GABP mAb in combination withprotein G-Sepharose, and analyzed by SDS-PAGE. The Western blot filterwas incubated with the anti-GANP mAB, followed by HRP-anti-rat Ig. Thedevelopment was performed using an ECL detection kit (Amersham).

(14) Reverse Transcriptase-PCR (RT-PCR)

Total RNA (1 μg each), purified from cultured B cells using TRISOL(Gibco BRL, Rockville, Md.) was used as a template for the cDNAsynthesis (100 μl volume) with Superscript (Gibco BRL). PCRamplification was carried out using 2 μl of each cDNA solution withTaq-Gold (Perkin-Elmer, Foster, Calif.) and the primers for ganp or HPRT(control) (Han, S., et al, 1996, Science. 274-2092-2097). The ganptranscripts were amplified by 5′-CCGTGGGATGACATCATCAC-3′ (the forwardprimer) (SEQ ID No. 5 of the sequence listing) and5′-CATGTCCACCATCTCCAGCA-3′ (the reverse primer) (SEQ ID No. 6 of thesequence listing).

<Results>

(1) Expression of the GANP Antigen in Lymphoid Organs

An mAb that recognizes a differentiation antigen expressed in peripheralB cells was prepared by immunizing rats with the lysate of WEHI-231cells. Immunohistochemical analysis with the 29-15 mAb on normallymphoid organs of BALB/c mice did not detect expression in the bonemarrow, but showed the slight expression in lymphoid organs such as thethymus, spleen, and lymph node. A small number of cells in the red pulpof the spleen and the deep cortex of the lymph node strongly express the29-15 Ag. Interestingly, the expression was very high in the centralarea of follicles of the PP (FIG. 1). The cells were positive withanti-B220 mAb, but not with anti-lgD mAb. Normal mice show thedevelopment of secondary lymphoid follicles with clear GC in PP becauseof the continuous stimulation of various antigenic substances introducedthrough the intestinal lumen.

Repeated immunization with sheep red blood cell (SRBC) induces theformation of lymphoid follicles in the spleen within 12 days. Antigenimmunization induces an appearance of 29-15⁺ cells in the GC area of thespleen and lymph node as well as in the GC of the PP (FIG. 2). The 29-15antigen appeared upregulated in cells of the GC. The phenotype of 29-15⁺cells in the architecture of secondary lymphoid follicles was furtheranalyzed. Nearly half of PNA⁺ GC-B cells are positive with the 29-15mAb, but they are negative with anti-BrdU mAb (FIG. 3). Interestingly,the expression of 29-15 Ag is upregulated in the centrocyte area at thedistal region of the entrance from the central artery. This phenotype isconsistent with the criteria of GC-B cells and supports the name “GANP”for the 29-15 Ag as described above.

(2) Appearance of GANP^(dense+) B Cells in the Red Pulp-Area ofAutoimmune-Prone NZB Mice

Normal mice express few GANP⁺ B cells in the follicular area of thespleen without in vivo stimulation but show a few GANP^(dense+) cellswhich remarkably express GANP protein in the red pulp area of BALB/c(FIG. 2) and C57BL/6. These cells are large and obviously different fromconventional B cells. In young (8 week old) NZB mice, however, theseGANP^(dense+) cells increased spontaneously in the red pulp area of thespleen without immunization (FIG. 4). Another autoimmune-prone mouse,NZW, does not express GANP^(dense+) cells in the red pulp at ages of 5to 12 weeks. A severe-disease combination of (NZB×NZW)F₁ shows anintermediate expression of GANP^(dense+) cells in the red pulp.

Whether the GANP^(dense+) cells also appear spontaneously in the spleenof other autoimmune-prone mice was examined. The GANP^(dense+) cellsappear in the spleen of BXSB and MRL/lpr, but not markedly in NZW andNOD mice at a similar age in the specific pathogen free condition (SPF).The GANP^(dense+) cells become apparent during aging and appear in theperipheral lymph node of the aged-NZB mice (10 month old) that havepassed the onset of the disease. The appearance of the GANP^(dense+)cells in the lymph node seems to be mostly in the later stage. Ofparticular interest, MRL/lpr shows the appearance of GANP^(dense+) cellsin the lymph node at the young stage (8 week old)(FIG. 5). These resultssuggested that a genetic factor in autoimmune-prone NZB, BXSB, andMRL/lpr mice might control the appearance of GANP^(dense+) cells in thered pulp area and the recruitment into the lymph node.

Two-color analysis showed the phenotype of GANP^(dense+) cells in thered pulp area as PNA⁻B220⁻ cells (FIG. 6) and IgD-CD38⁻ cells. Thesecells are positive when stained with anti-Syndecan-1 mAb, which stainsplasma cells selectively. The GANP^(dense+) cells express IgM incytoplasm (FIG. 7). Because these cells could be Mott cells (Jiang, Y.,S. Hirose, Y. Hamano, S. Kodera, H. Tsurui M. Abe, K. Terashima, S.Ishikawa and T. Shirai. 1997. J. Immunol. 158:992-997.), the section wasstained with PAS staining. The GANP^(dense+) cells show PAS⁻, as withthe B220-Syndecan-1⁺PNA⁻ BrdU⁻GANP^(dense+) (FIG. 8) and CD40⁻CD38⁻.These plasma-like cells appear preferentially in the spleen of NZB mice,but are different from Mott cells currently reported .

(3) Identification of a cDNA Clone Encoding the GANP Antigen

Using the 29-15 mAb, we isolated a candidate cDNA clone (with the insertDNA of 280 bp) from the WEHI-231 cDNA library and further isolated alonger cDNA clone, named ganp. The full-length nucleotide sequence (6429bp) determined from overlapping clones shows a putative polypeptidecomposed of 1971 amino acids with a predicted molecular size of 210-kD(FIG. 9). The amino acid sequence of GANP protein is shown in SEQ ID No.1 of the sequence listing and the base sequence of ganp cDNA is shown inSEQ ID No. 2 of the sequence listing.

The GANP amino acid sequence shows a regional homology to SAC3 which isconsidered to be a nuclear transcription regulation factor characterizedin temperature-mutant Saccharomyces cerevisiae and human Map 80 protein(Takei, Y et al,. 1998, J. Biol. Chem. 273:22177-22180) (FIG. 10 andFIG. 15; Bauer, A. and R. Koelling. 1996. Yeast 12:965-975). The GANPprotein shows mild homologies within short stretches of the insulinpromoter factor (amino acids 996 to 1063) and various transcriptionfactors, including NF-IL-6 (amino acids 388 to 450).

The GANP gene shows a consensus base sequence for the super coil motifs,but does not show zinc-finger, leucine-zipper, and homeo-domain motifs.A serine/threonine-rich region was seen in N-terminal 100 amino acids,which has slight homology to nucleoporin, which is known as the nuclearpore complex. GANP has two possible nuclear localization sequences(⁴⁹⁷HKKK and ¹³⁴⁴PMKQKRR), which would potentially support theexpression of the GANP in the nucleus as suggested by the PSORT program.Moreover, GANP has 2 coiled-coil motifs, but does not have zinc-finger,leucine-zipper, and homeo-domain motifs. Further, there were 4 LXXLLmotifs which were recognized in nuclear transcription coactivatormolecules including CBP/p300 and p/CIP (Torchia, J. et al., 1997. Nature(Lond.) 387:677-684; Heery et al., 1997, Nature (Lond.) 387:733-736),but any association molecule through these motif. has not beenidentified.

(5) Expression of the Ganp Transcripts

Northern blot analysis detected the 7-kb mRNA as a very weak signal incomparison to the control β-actin signal, but its expression was ratherubiquitous in all cell lines, organs, and tissues tested. In order toexamine whether the ganp mRNA is upregulated in the same areas asdetected on sections with the 29-15 mAb, in situ RNA hybridizationanalysis was carried out. The ganp mRNA is expressed abundantly in thecentral area of the GC of the SRBC-immunized spleen, but not in thenonimmunized spleen (FIG. 11), thymus, and lymph node. The ganp mRNA wasupregulated in GC-B cells of immunized mice. This expression pattern isquite similar to the results with the 29-15 mAb on the same sectionbased on staining with hematoxylin. The GC area of the PP also showedupregulation of the ganp mRNA in noninmnunized BALB/c mice, and theexpression of ganp mRNA is high in plasma-like cells of the red pulparea of the spleen of nonimmunized NZB mice (FIG. 11). These resultssuggests that the ganp gene encodes a molecule recognized by the 29-15mAb.

(6) Expression of the GANP in B Cells

The anti-GANP mAb (42-23) detected a single protein band at 210-kD fromboth nuclear and cytoplasmic compartments of WEHI-231 cells (FIG. 12).In order to find evidence of the functional involvement of the GANP inthe activation and differentiation of B lineage cells, B cells fromnonimmunized BALB/c mice were stimulated in vitro with anti-lgM andanti-CD40 in combination, and as a result, an expression of the GANPprotein detected with the anti-GANP mAb was increased (FIG. 13). An invitro kinase reaction with the GANP immunoprecipitates showed anincreased kinase activity assembled with the GANP protein in spleen Bcells stimulated in vitro. Thus, the GANP protein is induciblyphosphorylated at the serine/threonine residues (FIG. 14). These resultssuggest that the GANP might play a role to the activation of B cells inperipheral immune responses.

Stimulation with anti-μAb and anti-CD40 mAb showed maximal response, buteither one of these regents showed only a marginal response (data notshown). This upregulation was also detected by the increase of ganp mRNAin B cells stimulated by anti-μ and anti-CD40 co-ligation in vitro (FIG.16). RT-PCR clearly demonstrated that the amount of ganp mRNA increasedat 24 hours and 48 hours after stimulation in comparison with thecontrol HPRT mRNA.

Since the 210-kDa GANP has many possible phhosphorylation sites, weexamined the induction of phosphorylation by an in vitro kinase reactionwith anti-GANP immunoprecipitates. As shown in FIG. 17, phosphorylationof the 210-kDa protein was found in the anti-GANP immunoprecipitatesfrom spleen B cells stimulated by anti-μ and anti-CD40 co-ligation. Thisresult indicates that a kinase activity is maintained even if GANP isprecipitated.

(7) Association of GANP with MCM3 Protein

We found a Map80-homologous region (76.3% identity at amino acid level)in the carboxyl-terminal part of GANP. Map80 is an 80-kDa nuclearprotein that is involved in the translocation of MCM3 (a proteinessential for DNA replication) between the cytoplasm and the nuclei(Takei, Y. et al, 1998, J. Biol. Chem. 273:22177-22180; Kimura, H. etal, 1994, EMBO J. 13:4311-4320; Chong, J. P. et al, 1996, Trends.Biochem Sci. 21:102-106; and Romanowski, P et al, 1996, Curr. Biol.6:1416-1425). Therefore, we examined the interaction between GANP andMCM3 in WEHI-231. We detected that anti-GANP immunoprecipitates includeMCM3. Because the phosphorylation states of MCM proteins seem crucial inregulation of cell cycle progression (Kimura, H. et al, 1994, EMBO J.13:4311-4320; Chong, J. P. et al, 1996, Trends Biochem Sci. 21:102-106;and Romanowski, P et al, 1996, Curr. Biol. 6:1416-1425), in vitro kinaseassays with anti-MCM3 immunoprecipitates was performed.Immunoprecipitation of MCM3 co-precipitated a phosphorylated proteinmigrated at 210-kDa, which is the identical size of GANP (FIG. 19, leftpanel). These 210-kDa bands from anti-GANP and anti-MCM3immunoprecipitates showed an identical pattern in the V8 cleavagemapping (FIG. 19, right panel), indicating that GANP and MCM3 areassociated in a B cell line.

Next, we studied whether MCM3 is upregulated in GC-B cells byantigen-immuniization of mice in vivo. The contiguous sections to thoseused above were stained with the anti-MCM3 Ab (FIG. 20). MCM3 is alsoupregulated in GCs. Double staining clearly demonstrates theco-localization of both MCM3 and PNA. A part of GC area is surroundedintensely with FDCs (lymph follicular cells). These results demonstratethat MCM3 is upregulated in GC-B cells including centroblasts and theGANP⁺ centrocytes that would be mostly surrounded by FDCs (FIG. 20).

(8) Discussion

As mentioned above, the present inventors found a novel protein, GANP,expressed in GC-B cells localized at the light zone of secondaryfollicles in the spleen. Although a trace amount of the ganp mRNA isdetectable in many kinds of cells under normal conditions, the GANPprotein appears upregulated in the specified GC area of inmunized mice.A number of studies demonstrated various differentiation antigens in theGC as molecules recognized with mAbs or by specific cDNA cloning(Christoph et al., Int. Immunol. 6:1203-1211, 1994; Li et al., Proc.Natl. Acad. Sci. USA. 93:10222-10227, 1996; Kuo et al., J. Exp. Med.186:1547-1556, 1997). Most molecules appear in GC-B cells of the wholearea, whereas 8-oxoguanine DNA glycosylase is expressed in the dark zone(Kuo et al., J. Exp. Med. 186:1547-1556, 1997).

Interestingly, the GANP antigen is selective in the centrocyte of thelight zone. Recent studies have shown that RAG protein which isnecessary for rearrangement of immunoglobulin gene is selectivelyexpressed in centrocytes at the light zone (Hikida et al., 1996. Science(Wash. D.C.) 274:2092-2094, 1996; Han et al., Science (Wash. D.C.)274:2094-2097, 1996). Since the GC area-probably provides the site forsecondary lg gene rearrangement occurring during T cell-dependentantibody responses, as described by Papavasiliou et al. and Han et al.(Papavasiliou et al., Science (Wash. D.C.), 278:298-301, 1997; Han etal., Science (Wash. D.C.), 278: 301-305, 1997), the GANP protein mightbe a component associated with the maturation of antigen-specific Bcells at the centrocyte stage.

We found that the carboxyl-terminal portion of GANP has a significantsimilarity to human Map80, which facilitates the nuclear transport ofMCM3 (Takei, Y et al., 1998, J. Biol. Chem.273:22177-22180).Immunoprecipitation experiments demonstrated that GANP also binds toMCM3 in WEHI-231. MCM3 is a member of the MCM protein family essentialfor the initiation of DNA replication (Kimura, H. et al, 1994, EMBO J.13:4311-4320; Blow, J. J. 1993. J. Cell Biol. 122.993-1002; Tye, B. K1994. Trends Cell Biol. 4: 160-166; Chong, J. P. et al, 1996, TrendsBiochem Sci. 21:102-106; Romanowski, P et al, 1996, Curr. Biol.6:1416-1425; and Thommes, P et al, 1992, Nucl. Acids Res. 20:1069-1074). The major fractions of nuclear MCM proteins bind tochromatin at the beginning of the S phase, but dissociate duringreplication and accumulate as free proteins in the nucleosol. Therelease of MCMs from chromatin is accompanied by the phosphorylation ofseveral MCM proteins and their reassociation after mitosis isconcomitant with their dephosphorylation. It was suggested that MCMproteins are no longer synthesized in growth arrested, differentiatingcells and disappear with kinetics related to their half-life (Musahl,C., et al, 1998, Exp. Cell. Res. 241, 260-264). The MCM3 protein hasrecently been shown to an early target in apoptotic proteolysis (Schwab,B. L. et al., 1998, Exp. Cell Res. 238:415-421). Schwab, B. L. et alproposed that active destruction of MCM3 inactivates the MCM complex andserves to prevent untimely DNA replication events during the executionof the cell death program. Our results showed that GC-B cells expresshigh level of MCM3, some of which is associated with GANP. However, itappears curious that a protein, upregulated in differentiated cells thatarrest the cell cycle, binds to another protein essential forprogression of the S phase. One possible speculation is that a functionof GANP may be inactivation of MCM3 through its binding. Theimmunohistochemistry data are consistent with the following idea; GANPis upregulated in growth-arrested centrocytes while MCM3 is expressedboth in rapid-cycling centroblasts and still in centrocytes in GCs.Although the amount of MCM3 would decrease by ceasing the geneexpression and active destruction (Musahl, C., et al, 1998, Exp. Cell.Res. 241, 260-264; and Schwab, B. L. et al., 1998, Exp. Cell Res.238:415-421), inactivation of MCM3, which is still expressed incentrocytes, through the interaction with GANP could be anothermechanism to prevent DNA replication. In addition, both GANP and MCM3become phosphorylated with the co-precipitated kinase (FIG. 19). Sincethe highly phosphorylated MCM3 is thought to be inactivated form(Kimura, H. et al, 1994, EMBO J. 13:4311-4320), the association withGANP may stimulate phosphoryltion of MCM3.

The GANP protein has a close similarity to the SAC3 (SAC, suppressor ofactin) of yeasts, Saccharomyces cerevisiae, which was isolated in agenetic screen for suppressors of a temperature-sensitive mutation(act1-1) in the actin gene (FIG. 10; Novick et al., Genetics,121:659-674, 1989). The SAC3 protein is expressed in the nuclei and isrequired for normal progression of mitosis and protection against theloss of chromosomes (Bauer et al., J. Cell. Sci. 109:1575-1583, 1996).Null mutants of SAC3 grow very slowly and are larger than wild-typecells. SAC3 participates in a process that affects both the actincytoskeleton and mitosis, which suggest that SAC3 regulates the geneexpression of actin or actin-binding proteins.

A gene (named LEP-1) that augments the transcription of the leucinepermease activity in Saccharomyces was identical to SAC3 (Stella et al.,Yeast 11:460-460, 1995). Although the LEP-1 gene induces theupregulation of the yeast leucine permease involved in selective aminoacid transport, the amino acid transport in eukaryotic cells, especiallythe molecules involved in amino acid permeation is not known(Mastroberardino et al., Nature (Lond.) 395:288-291, 1998). Although theSAC3/LEP-1 sequence does not show motifs homologous to a number oftranscription factors, the biological functions determined previously(Bauer et al., J. Cell. Sci. 109:1575-1583, 1996) suggest its regulatoryactivity of various target genes in the nucleus. The mouse GANP does notshow typical consensus motifs for nuclear transcription factors, but hasa common ancestor with SAC3 gene of yeasts and has structural similarityof possible phosphorylation sites, two nuclear localization sequences,and two super coil structures that might interact with othertranscription molecules.

GANP is selectively upregulated in centrocytes of Ag-immunized spleen.It is also useful as the differentiation marker to define the centrocytesubset that is closely interacting with FDCs in GC area. Our studyshowed that the BCR signal and the CD40 co-stimulation together causethe upregulation of GANP and lead to the signal transduction mediatedthrough GANP/MCM3 complex.

The defective gene in the autosomal recessive genetic disease autoimmunepolyendocrinopathy (APECED) is localized by linkage analysis to humanchromosome 21 (21q22.3), which encodes an AIRE gene product with apossible transcription regulator (Nagamine et al., Nature Genet.17:393-398, 1997). The autoantibody recognizes the AIRE proteinexpressed in the adrenal gland and other gonad-producing tissues.Studies of APECED drew an idea that the involvement of molecules withnuclear coactivator activity might be associated with the autoimmunity.Both the AIRE and GANP proteins do not have typical domains fortranscription regulators, but they have LXXLL motifs as similarlyobserved in nuclear transcriptional coactivators.

A B cell-specific nuclear coactivator (Bob1/OCA-B/OBF1) was recentlycharacterized as a cell-type-specific regulator of Oct1 and Oct2 (Luo etal., Mol. Cell. Biol. 15:4115-4124, 1995). The OCA-B targeted mice showthe impairment of the GC formation in the spleen after immunization withT-dependent antigen, which suggests the functional involvement of B cellmaturation in the GC area (Kim et al., Nature (Lond.) 383:542-547, 1996;Qin et al., EMBO J. 17:5066-5075, 1998). The expression of the GANPprotein might be under the control of the OCA-B cell in centrocytes. Themolecular interaction of the nuclear coactivator molecules would be animportant issue for the understanding of the B cell maturation in theGC.

The New Zealand model of SLE has been the experiment subject of genomelinkage studies to map the chromosomal positions ofdisease-susceptibility genes. At least 12 non-MHC loci linked withnephritis and autoantibody production such as on chromosome 4(designated Nba1), on chromosome 7, and on chromosome 1 (designated asNba2; Vyse et al., J. Immunol. 158:5566-5574, 1997) have beenindependently mapped. The GANP antigen on large cells is highlyupregulated in the red pulp area of the nonimmiunized NZB mice (FIGS.4-8). NZB mice contained similar large lgM-producing cells, named Mottcells, in the red pulp area. Mott cells appear selectively in NZB and(NZB×NZW)F₁ mice, but not in normal BALB/c or C57BU/6 mice.

The precursor cells of Mott cells are probably B-1 B cells (Tarlinton etal., Eur. J. Immunol. 22:531-539, 1992; Jiang et al., J. Immunol.158:992-997, 1997), which suggests a close association with theautoimmunity of B cells. Mott cells are apparent with the inclusion bodyof lgM in the cytoplasm and positive staining with PAS (Tarlinton etal., Eur. J. Immunol. 22:531-539, 1992; Jiang et al., J. Immunol.158:992-997, 1997). Because GANP^(dense+) cells seem to be Mott cells,PAS staining was performed. However, GANP^(dense+) cells in the red pulparea of NZB mice are PAS⁻. The GANP^(dense+) lgM-producing cells appearin the spleen of NZB mice, as do Mott cells, but these cells aredifferent. The new type of lgM-producing cells could be generated by thepossible activation of an abnormal B cell population related to one ofthe chromosomal loci linked to disease-susceptibility.

Lyn^(31/−) mice and CD40L^(−/−) mice reported from several laboratoriesshow similar autoimmunities and hyper-lgM syndrome(s), which have anincreased appearance of immunoblast cells with the inclusion body in thespleen (Hibbs et al., Cell 83:301-311, 1995; Nishizumi et al., Immunity3:549-560, 1995; Xu et al., Immunity 1:423-431, 1994). Theseobservations suggest that the signal transduction through BCR and CD40is regulating the generation of the abnormal antibody-producing plasmacells. Stimulation of splenic B cells with anti-lgM and anti-CD40antibodies induces the phosphorylation activity of the GANP protein.This observation suggests that the GANP protein may be involved indownstream of the B cell activation site in the GC area and the abnormalB cell activation in NZB mice might be associated with the increasedexpression of GANP protein.

Example 2 Cloning of Human GANP Gene

On the basis of information of the sequence of rat GANP gene, human GANPgene was cloned and sequenced. Specifically, λgt 11-human heart cDNAlibrary (Clontech) was used, and gsp1-1: TTTGTCTGGAGGATGATCGC (SEQ IDNo.7 of the sequence listing), gsp1-2: AAAGAGAAAGGGGCCAGGCC (SEQ ID No.8of the sequence listing) and gsp1-3: CCAGCTTCTTGTCCAAAAGC (SEQ ID No.9of the sequence listing) were used as primers, and 5′ RACE System forRapid Amplification of cDNA Ends, Version 2.0(Gibco BRL) was used tocarry out the cloning and sequencing by a conventional method.

The base sequence of the obtained clone was determined. The basesequence of the obtained human GANP gene is shown in SEQ ID No.4 of thesequence listing. The amino acid sequence encoded by this base sequenceis shown in SEQ ID No.3 of the sequence listing and FIG. 21. Human GANPgene shows high homology with mouse GANP gene, and Human GANP containsMap80 domain of 80 kDa at carboxyl terminal.

In in situ RNA hybridization, ganp transcript seems to be activated atGC region of tonsil. GANP⁺ cells express CD38⁺IgD⁺ phenotype of memory Bcell. These results show that human GANP is expressed also in GC-B cellsof secondary lympho tissues. Moreover, since human GANP of 1980 aminoacids has a stretch of Map80 homologous region which binds to MCM3protein in B cells, it is suggested that GANP is involved in theregulation of cell cycle in GC-B cells.

Furthermore, in situ hybridization was carried out by FISH method withthe obtained human GANP gene and human chromosome specimen. The resultsare shown in FIG. 22. As is understood from FIG. 22, the genome fragmentcontaining human GANP gene and Map80 was mapped on 22.3 of the long armof chromosome 21.

INDUSTRIAL APPLICABILITY

The protein of the present invention is a novel protein having a kinaseactivity, and may be involved in a signal conversion of abnormal B celldifferentiation in an autoimmune state. Therefore, the protein,polypeptide, polynucleotide, antisense polynucleotide and antibody ofthe present invention are useful for revealing the mechanism ofautoimmune.

1-9. (canceled)
 10. A polynucleotide which encodes a protein having theamino acid sequence shown in SEQ ID No.
 1. 11. An antisensepolynucleotide, which comprises the base sequence of an antisense chainof the polynucleotide of claim
 10. 12. A polynucleotide which encodes avariant protein having a kinase activity substantially similar to aprotein having the amino acid sequence shown in SEQ ID No. 1, wherein1-20 amino acids are deleted, substituted, and/or added.
 13. Anantisense polynucleotide, which comprises the base sequence of anantisense chain of the polynucleotide of claim 12.